In the recent years, the research in the biosensor vigorously proceeds because of the development of the nanotechnology, the miniaturization of the biochemical medical sensor and the demands on low concentration and high precision. Please refer to FIG. 1, which is a schematic diagram showing a conventional biosensor 10. The biosensor 10 includes an analyte layer 101, a biorecognition element layer 102 and a biotransducer 103. In general, the analytes in the analyte layer 101 is carried in the fluid 104 flowing to pass the biorecognition element layer 102. A mechanism is used to bind the analytes to the biorecognition element layer 102 for recognizing the analytes. The mechanism may be the configuration complementation between the antibody and the antigen, or at least one of the ionic bond, the hydrogen bond, the Van Der Waals' force and the hydrophobicity gravity between molecules. The configuration complementation may be the inter-binding mechanism between two protein molecules, that is, the shapes of the lock and the key must be complementary in order to bind them.
Please refer to Table 1, which is a table showing types of biosensors, and recognition mechanisms thereof The biosensors may be classified as a direct bioaffinity biosensor and a biocatalytic sensor. The corresponding relations among the signal generating method, the bioaffinity-corresponding object and the analyte for the biosensors are shown in Table 1.
TABLE 1Bioaffinity-Signal generatingcorrespondingSensor typemethodobjectAnalyteDirectDirectly generatingEnzymeSubstratebioaffinitybonding between theanaloguebiosensorbiorecognitionAntibodyAntigenelement layer and theVirusanalyte layerCellNucleic acidComplementarysequenceLectinGlycoproteinBiocatalyticTransforming toChemicalTarget analytesensorgenerate metabolitereceptorApoenzymeProsthetic groupEnzymeInhibitorAntibodyEnzyme-markedantigenChemicalEnzymeSubstratetransformationOrganelleCofactorMicrobe
Types of biosensors may be classified as an optical type, an electrochemical type and a mechanical type according to the signal generating methods thereof. At present, the optical-type biosensor is widely used. However, it is uneasy to carry the optical-type biosensor because the volume thereof is too big.
The mechanical-type biosensor estimates the weight variation by measuring the resonance variation resulting from binding the analyte and the reactant. However, it is easy to result in bad sensitivity because of the influence of the structure on the mechanical-type biosensor itself. Therefore, the research focus is turned to the piezoresistive-type microcantilever.
The piezoresistive-type microcantilever mainly uses the influence of the stress on it to detect the analyte. When the analyte is absorbed on the piezoresistive-type microcantilever, the surface stress thereof can be changed because of the action force among the molecules, which causes the piezoresistive-type microcantilever to bend upward or downward, so that the piezoresistance thereof is changed. The action force among the molecules involves the mutual-absorption or the mutual-repulsion of the electrostatic force, the pushing effect resulting from the space limit among the molecules, the hydrophily/hydrophobicity change on the surface of the piezoresistive-type microcantilever, the configuration change of the absorbed biomolecules, and the change of the environmental solution.
The piezoresistive-type microcantilever includes a piezoresistor having a piezoresistance. The piezoresistive-type microcantilever has a disadvantage due to a resistance temperature coefficient effect and a bimorph effect both resulting from the temperature variation of the piezoresistive-type microcantilever or the environmental temperature variation. The resistance temperature coefficient effect results from that the resistance temperature coefficient of the piezoresistor itself is changed under the influence of the temperature variation of the piezoresistive-type microcantilever, and results in the variation of the piezoresistance thereof, and such the piezoresistance variation is known as the resistance temperature coefficient effect of the piezoresistive-type microcantilever. In general, the bimorph effect happens in a piezoresistive-type microcantilever including a plurality of layers made of a multilayer composite material, wherein the plurality of layers have different thermal expansion coefficients. When the environmental temperature or the temperature of the piezoresistive-type microcantilever varies, the differences among the expanded lengths of the plurality of layers result in that the piezoresistive-type microcantilever is acted under stress to bend upward or downward, which causes the variation of the piezoresistance thereof. The piezoresistance variation resulting from the temperature variation is the main reason resulting in an error, so that many methods to reduce the error are proposed.
Please refer to FIG. 2(a), which is a schematic diagram showing a conventional microcantilever sensing apparatus 20. The microcantilever sensing apparatus 20 includes a reference microcantilever 201, a sensing microcantilever 202 and a Wheatstone bridge 203. The Wheatstone bridge 203 includes resistors A1, A2, A3 and A4 respectively having resistances R1, R2, R3 and R4. The Wheatstone bridge 203 receives an input voltage VIN. The voltage V13 comes from distributing the input voltage VIN to the resistances R1 and R3. The voltage V24 comes from distributing the input voltage VIN to the resistances R2 and R4. There is the voltage VOUT=V13−V24.
The reference microcantilever 201 includes a reference resistor A21 serving as the resistor A2. The reference resistor A21 has a reference resistance R-ref; that is, the reference resistance R-ref is the resistance R2. The sensing microcantilever 202 includes a sensing resistor A22 serving as the resistor A1. The sensing resistor A22 has a sensing resistance R-sensor; that is, the sensing resistance R-sensor is the resistance R1. The variation of the reference resistance R-ref and that of the sensing resistance R-sensor are considered as follows. According to the voltage-dividing theorem, the voltage V13 satisfies V13=VIN×R3/(R1+R3) and the voltage V24 satisfies V24=VIN×R4/(R2+R4). Therefore, the voltage VOUT satisfies VOUT=V13−V24=VIN×[(R3/(R1+R3))−(R4/(R2+R4))]. In one practical application, the resistances R1, R2, R3 and R4 satisfy R1=R2=R3=R4=R. When only the resistance R1 has a tiny variation ΔR1, the voltage VOUT satisfies VOUT=VIN×[(R/(ΔR1+R+R))−(R/2R)]. Therefore, the voltage VOUT satisfies VOUT≈VIN×(−ΔR1/4R) because of ΔR1<<R. Here, a voltage VOUT1 is used to represent the output voltage resulting from the tiny variation ΔR1. As a result, when the resistance R1 has the tiny variation ΔR1, the tiny variation ΔR1 is converted into the voltage VOUT. An amplifier (not shown) is further used to amplify the voltage VOUT so that a variation of the voltage VOUT may be measured.
Similarly, when only the resistance R2 has a tiny variation ΔR2, the voltage VOUT satisfies VOUT=VIN×[(R/2R)−(R/(ΔR2+R+R))]≈VIN×(ΔR2/4R). Here, a voltage VOUT2 is used to represent the output voltage resulting from the tiny variation ΔR2. Preferably, the sensing resistance R-sensor is the same to the resistance R1 and the reference resistance R-ref is the same to the resistance R2. In theory, if the reference microcantilever 201 and the sensing microcantilever 202 have the same structure and the same material, when the environmental temperature varies and the tiny variations ΔR1 and ΔR2 satisfy ΔR1=ΔR2, the voltages VOUT1 and VOUT2 can neutralize each other.
In one practical application, the material deposited on the sensing microcantilever 202 is different from that deposited on the reference microcantilever 201 due to different measuring functions. Therefore, when the environmental temperature varies and the tiny variations ΔR1 and ΔR2 satisfy ΔR1≠ΔR2, the bimorph effect of the reference microcantilever 201 and that of the sensing microcantilever 202 can affect the detection precision. Besides, if the acid-base concentration, such as the pH value, of the analyte solution varies, it is possible to result in the condition that the voltage VOUT1 is out of phase with the voltage VOUT2, which interferes the interpretation of the produced real signal.
Please refer to FIG. 2(b), which is a schematic diagram showing voltages obtained from the conventional microcantilevers with pH values of an analyte solution. In FIG. 2(b), the time when an analyte solution reacts with the sensing microcantilever 202 and the time when the analyte solution reacts with the reference microcantilever 201 are expressed in the abscissa axis, and the unit in the abscissa axis is the minute. The unit in the ordinate axis is the volt. The hollow circles denote the voltage points obtained by measuring with the sensing microcantilever 202. The solid circles denote the voltage points obtained by measuring with the reference microcantilever 201. It can be seen in FIG. 2(b) that the pH value of the analyte solution is gradually changed from a smaller value to a larger value with time. When the time reaches the time point of 260 minutes, the pH value of the analyte solution is 12 and the voltage obtained by measuring with the sensing microcantilever 202 is out of phase with the voltage obtained by measuring with the reference microcantilever 201, so that what the produced real voltages mean cannot be interpreted.